Nowadays, alkylating anticancer agents, as nitrogen mustards, still remain an effective class of antitumor drugs in current clinical practice, whose therapeutic effects derive from their ability to attach alkyl groups to cellular DNA and to produce significant DNA damage (Hurley L H, Nature Rev Cancer, 2002, 2:188-200; Brendel M and Ruhland A, Mutat Res, 1984; 133:51-85).
Steroidal conjugates have been previously used as carriers of cytotoxic alkylating agents because they reduce systemic toxicity and improve efficacy of cancer therapy (Wall M E et al, J Med Chem, 1969, 12:810-8; Catane R, Cancer Treat Rep, 1978; 62:1264-5). Steroidal alkylating agents as Estramustine (ester of estradiol and mechlorethamine) and Prednimustine (ester prednisolone and chlorambucil) are currently applied in cancer therapy on the treatment of prostate cancer and lymphoproliferative malignancies respectively (Catane R, Cancer Treat Rep, 1978, 62:1264-5; Matsumoto K et al, Med Oncol, 2013, 30:717; IARC Monogr Eval Carcinog Risks Hum, 1990, 50:115-22; Hiddemann W, Eur J Cancer, 1995, 31A (13-14):2141-5).

Whereas these drugs produce diminished acute and systemic toxicity contrary to the much higher toxicity that their alkylating components produced alone, their anticancer activity is not so much improved as well as specificity to targeting cancer cells rather be short despite the initial evaluations. However, even if the main molecular pharmacological mechanisms that estramustine and prednimustine exert anticancer activity are rather different than specific action on the steroid receptors, in general they showed good and improved therapeutic efficacy in clinical practice.
Several homo-aza- or lactam steroidal esters (steroids that contain lactam group —NHC═O— into steroid ring/s conjugated with alkylating agents) have been previously synthesized and tested for toxicity and anticancer activity in preclinical settings, in vitro and in vivo (Wampler G L and Catsoulacos P, Cancer Treat Rep, 1977, 61:37-41; Catsoulacos P and Catsoulacos D, Anticancer Res, 1991, 11:1773-7; Catsoulacos P and Catsoulacos D, Anticancer Res, 1993, 13 (4):1203-8; Catsoulacos P et al, Oncology, 1994, 51:74-8; Catsoulacos P and Catsoulacos D, Anticancer Res, 1994, 14 (6B):2525-8; Camoutsis C and Trafalis D T, Invest New Drugs, 2003, 21:47-54; Koutsourea A I et al, Bioorg Med Chem, 2008, 16:5207-15).
Lactam steroid alkylating esters showed that they generate significantly decreased acute toxicity in vivo, whereas they demonstrated enhanced and very promising antitumor activity in vitro and in vivo, while the respective unmodified (non-lactam) steroidal alkylators produced significantly lower or little anticancer activity against the respective experimental tumor systems. Except of the production of cellular DNA damage, the molecular pharmacological mechanisms that significantly improved anticancer effect of the lactam steroid alkylating esters take action are still uncharted. Moreover, the biological importance of the position that one or more lactam groups are incorporated into the steroidal structure is also unknown. Furthermore, the alkylating agent that conjugated via esteric bond on the lactamic steroid plays significant role and modulates the proportion of acute toxicity and antitumor activity, and consequently the extent of therapeutic ratio that a lactam steroidal alkylator generates. Up to now, several active lactam steroidal alkylators have been synthesized and tested but those which showed higher antitumor activity were more toxic and those which demonstrated lower toxicity were less active. These observations indicate that there is a clear need to develop and produce novel active lactam steroidal alkylating conjugates that generate optimal the lower toxicity and higher anticancer activity and therefore the optimum therapeutic index.
Previous studies on lactam steroidal alkylating esters of nitrogen mustard derivatives showed that 3beta-hydroxy-13alpha-amino-13,17-seco-5alpha-androstan-17-oic-13,17-lactam-[p-[bis (2-chloroethyl)amino]phenyl]acetate (ASE, NSC-290205) produced very well balanced effects in preclinical testing on acute toxicity in vivo and antitumor activity in vitro and in vivo, holding a significantly high therapeutic index.

Therefore ASE represented the “golden” standard for developing new molecules of the same class of agents and testing them for therapeutic efficacy in comparison to that of ASE.